Liposomes, or lipid bilayer vesicles, have been used or proposed for use in a variety of diagnostic and therapeutic applications. Particularly in their use as carriers in vivo of diagnostic or therapeutic compounds, the liposomes are typically prepared to contain the compound in liposome-entrapped form.
Ideally, such liposomes can be prepared to include the entrapped compound (i) with high loading efficiency, (ii) at a high concentration of entrapped compound, and (iii) in a stable form, i.e., with little compound leakage on storage.
Methods for forming liposomes under conditions in which the compound to be entrapped is passively loaded into the liposomes are well known. Typically, a dried lipid film is hydrated with an aqueous phase medium, to form multi-lamellar vesicles which passively entrap compound during liposome formation. The compound may be either a lipophilic compound included in the dried lipid film, or a water-soluble compound contained in the hydrating medium. For water-soluble compounds, this method gives rather poor encapsulation efficiencies, in which typically only 5-20% of the total compound in the final liposome suspension is in encapsulated form. Additional compound may be lost if the vesicles are further processed, i.e., by extrusion, to produce smaller, more uniformly sized liposomes. The poor encapsulation efficiency limits the amount of compound that can be loaded into the liposomes, and can present costly compound-recovery costs in manufacturing.
A variety of other passive entrapment methods for forming compound-loaded liposomes, including solvent injection methods and a reverse-evaporation phase approach (Szoka and Papahadjopoulos, 1978) have been proposed. These methods tend to suffer from relatively poor loading efficiencies and/or difficult solvent handling problems.
It has also been proposed to passively load compounds into liposomes by incubating the compound with preformed liposomes at an elevated temperature at which the compound is relatively soluble, allowing the compound to equilibrate into the liposomes at this temperature, then lowering the temperature of the liposomes to precipitate compound within the liposomes. This method is limited by the relatively poor encapsulation efficiencies which are characteristic of passive loading methods. Also, the compound may be quickly lost from the liposomes at elevated temperature, e.g., body temperature.
Compound loading against an inside-to-outside pH or electrochemical liposome gradient has proven useful for loading ionizable compounds into liposomes. In theory, very high loading efficiencies can be achieved by employing suitable gradients, e.g., pH gradients of 2-4 units, and by proper selection of initial loading conditions (Nichols and Deamer, 1976). With this method, compound leakage from the liposomes will follow the loss of ion gradient from the liposomes. Therefore, compound can be stably retained in liposome-encapsulated form only as long as the ion gradient is maintained.
This gradient stability problem was addressed, and at least partially solved, by employing an ammonium salt gradient for compound loading (Haran, et al., 1993). Here excess ammonium ions, which act as a source of protons in the liposomes, function in addition as a battery to replenish protons lost during storage, thus increasing the lifetime of the proton gradient, and therefore reducing the rate of leakage from the liposomes. The method is limited to ionizable amine compounds.
Lastly, the utility of precipitation for liposome loading was hypothesized in the literature. Lasic D. D. (1993).